The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of molybdenum nitrogenase and the iron-vanadium cofactor (FeV-co) of the vanadium nitrogenase will be investigated. These cofactors serve as the active sites in nitrogenases which carry out the ATP-dependent reduction of N2 to ammonium and provide the N-needed for synthesis of proteins, nucleic acids and other nitrogenous compounds in the cell. FeMo-co is the bette understood of the cofactors and consists of MoFe7S8-9homocitrate; the structure of FeMo-co has recently been determined. The iron-vanadium cofactor is thought to have a structure that is very similar to FeMo-co. The specific goals of this project are: 1) To determine the structure and the role of a recently identified precursor to FeMo-co. This FeS compound is produced by cells with an active nifB gene and is called NifB-co. NifB-co is required for the synthesis of both FeMo-co and FeV- co. 2) To develop an assay for the in vitro synthesis of NifB-co. 3) To determine the role(s) of the nifNE and vnfN gene products in FeMo-co and FeV-co synthesis, respectively. Our hypothesis is that NifNE and VnfNE serve as the scaffolds upon which FeMo-co and FeV-co are assembled 4) To determine the role(s) of the nifH and vnfH gene products (dinitrogenase reductase) in FeMo-co and FeV-co syntheses. Our hypothesis is that these gene products play a role in preparing NifNe an VnfNE proteins to bind NifB-co. The nifH and vnfH gene products may als play a role in specifying the metal (Mo or V) that is added to NifB-co. 5) To identify any additional components required for the synthesis of FeMo-co. There is strong evidence that FeMo-co synthesis requires components in addition to NifB-co, the NifNE protein, the NifH protein, homocitrate, molybdate, ATP and reductant. The in vitro FeMo-co synthesis assay will be used to identify those factors and will be used to follow the purification of the factors. The approach to the elucidation of this biosynthetic pathway will be to isolate the gene products and intermediates involved, characterize and identify them and, from this information, to deduce the pathway. Requirements for some of the steps are expected to be complex. Many of the components and intermediates of this pathway are oxygen-labile, and thus most of the isolations and characterizations will be performed using anaerobic techniques. While the ability to fix nitrogen is limited to a diverse set of procaryotic organisms, the nitrogen fixed is the ultimate N sourc for much of the life on earth. Nitrogenases contain complex metal clusters and the biosyntheses of these clusters is the goal of this project. Mo and Fe and important components of these clusters and these elements are essential for health of all organisms. The understanding of Mo, Fe and V processing that is gained in this project will be applicable to other systems as well.